1. Field of the Invention
The present invention relates to a method for monitoring differentiation into cardiac muscle cells.
2. Description of the Related Art
At present, regenerative medicine to regenerate affected parts or parts lost by diseases is drawing attention in the medical field. In the field of such regenerative medicine, attention is drawn to attempts to differentiate, into cardiac muscle cells, stem cells such as embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) having differentiation potency into various cells, and to use the cardiac muscle cells in the treatment of patients having diseases such as myocardial infarction.
However, many points are still unknown regarding the mechanism in which ES cells and iPS cells differentiate into various cells. Investigating how genes are expressed in the process of differentiation is extremely important in clarifying differentiation processes.
One method of analyzing gene expression is a technique of transcriptional activity measurement using reporter genes. The technique of transcriptional activity measurement includes an analysis of a gene expression control sequence such as a promoter, an enhancer, and a silencer, and transcription factors bound thereto. This technique also includes using the expression level of the reporter genes connected to a certain promoter as an index to analyze various intracellular molecular mechanisms, for example, to analyze the activation state of intracellular signaling pathways or analyze receptors and ligands. This technique is also used as a tool for large scale screening in drug discovery and as an index of toxicity assessment of chemical substances.
In general, techniques such as fluorescence imaging using fluorescent protein such as GFP, antibody staining, western blotting, flow cytometry and so on are used in the analysis of gene expression in a differentiation-inducing process of stem cells such as ES cells and iPS cells.
However, the problem of the fluorescence imaging is that quantitatively observing gene expression with time for a long period is difficult because of optical damage caused by excitation light, influence of autofluorescence, and a case where a desired amount of light may not reach due to the scattering of excitation light inside an embryoid body comprising cell groups that have differentiated. The problem of the antibody staining and the western blotting is that gene expression of the same target cannot be observed with time because a sample needs to be fixed.
Recently, not only fluorescence imaging using fluorescent protein but also luminescence imaging using bioluminescence of luminescent living organisms typified by fireflies has been performed. The bioluminescence is a phenomenon in which visible light is emitted by living organisms or substances derived therefrom due to various chemiluminescence reactions without dependence on optical energy resulting from illumination light. The bioluminescence reactions mostly require three components: luciferin (substrate), luciferase (enzyme), and molecular enzyme.
Luciferase is used as reporter genes for assessment of influence of exogenous factors on cells, intracellular information transmission, expression analyses of individual protein groups and so on. In a system in which luciferase is used, for example, firefly luciferase genes are first inserted into a transcriptionally activated site. A genetic construct is then introduced into cells. Cultured cells into which the reporter genes have been introduced are then treated with, for example, a drug for a predetermined time. The cells are then collected, and a luminescent substrate is added thereto. As a result, the amount of luciferase synthesized in the cells is measured, and transcriptional activity can be assessed.
Luminometers are generally used to assess the transcriptional activity of reporter assays using luciferase. However, the luminometers are intended to measure luminescence of all cells present in a well. Cells need to be dissolved depending on protocols. Therefore, the change of the luminescent intensity of each cell cannot be measured with time in the same cell in the assays using the luminometers.
In the utilization in the regenerative medicine of stem cells such as ES cells and iPS cells, it is reported that the degree and characteristics of gene expression vary from strain to strain. The challenge is therefore to adjust and maintain cells of uniform quality. It is thus necessary to clarify, on an individual single cell level, how genes are expressed in the differentiation-inducing process of ES cells and iPS cells.
Techniques to image stem cells such as ES cells and iPS cells are disclosed in International Publication No. 2011/029798 and International Publication No. 2007/080622. However, the techniques disclosed in International Publication No. 2011/029798 and International Publication No. 2007/080622 are techniques to measure the total of gene expression of stem cells by the luminometer or methods to transplant stem cells into a mouse body and then image the stem cells in the living body. That is, the techniques disclosed in International Publication No. 2011/029798 and International Publication No. 2007/080622 are not intended to image gene expression on an individual cell level.
A technique to monitor the differentiation of stem cells such as ES cells and iPS cells and the expression of genes involved in differentiation is disclosed in International Publication No. 2010/101225. The antibody staining method is used in the technique disclosed in International Publication No. 2010/101225. In this case, cells need to be fixed, and endpoint data alone are obtained. Moreover, fluorescence is used as a means of detection in the antibody staining. Cells having integrated structures such as a cell sheet or an embryoid body have strong autofluorescence. Therefore, a quantitative analysis using fluorescence is difficult in some cases.